The porcine reproductive respiratory syndrome virus (PRRSV) is the cause of a new porcine disease that has attacked over 5.000 North European pig farms since late 1990. This disease now called Porcine Reproductive Respiratory Syndrome (PRRS) is also known as Mystery Swine Disease. First, identified in Germany in December of 1990, the problem became increasingly critical in the beginning of 1991. In January and February of 1991 the disease spread to the Netherlands and Belgium. Outbreaks have also been reported from Spain. It is anticipated that the disease will become very costly from an economic standpoint, comparable to or even worse than Aujeszky's Disease.
The principal clinical signs in sows are anorexia and late abortion up to day 110 of pregnancy. With piglets a high incidence of stillborn and/or weak piglets in addition to respiratory problems are observed. In fatteners chronic pneumonia and increased mortality occur.
In order to develop a vaccine to protect pigs against PRRS or to develop a diagnostic method to determine infection in pigs, the causative agent of the disease has to be identified, isolated and made suitable as an immunogen in a vaccine or antigen in a diagnostic assay.
Conventional vaccines may comprise chemically inactivated virus vaccines or modified live-virus vaccines. However, inactivated vaccines require additional immunizations, disadvantageously contain adjuvants, are expensive to produce and are laborious to administer. Furthermore, some infectious virus particles may survive the inactivation process and cause disease after administration to the animal.
In general, attenuated live virus vaccines are preferred because they evoke an immune response often based on both humoral and cellular reactions. Up to now, such vaccines based on PRRSV strains can only be prepared by culturing and serial passaging of virulent strains in macrophage cell culture. However, because of this treatment uncontrolled mutations are introduced into the viral genome, resulting in a population of virus particles heterogeneous in their virulence and immunizing properties. In addition it is well known that such traditional attenuated live virus vaccines can revert to virulence resulting in disease of the inoculated animals and the possible spread of the pathogen to other animals. Furthermore, culturing of the PRRSV strain on a macrophage cell line is very laborious and results in a pour yield of the virus.
Improved vaccines, both live- and subunit vaccines, may be constructed based on recombinant DNA technology. These vaccines would only contain the necessary and relevant PRRSV immunogenic material capable of eliciting an immune response against the PRRSV pathogens, or the genetic information encoding said material, and not display above-mentioned disadvantages of the live or inactivated vaccines.
The causative agent of the disease is now known to be a small enveloped RNA virus, and the European type of this virus has been described by Wensvoort et al. (The Vet. Quarterly 13, 121-130, 1991). Its relatedness to Lactate Dehydrogenase-elevating virus (LDV) and Equine Arteritis virus has been described by Conzelmann et al. (Virology 193, 329-339, 1993), and by Meulenberg et al. (Virology 192, 62-72, 1993), thus placing the virus in the group of Arteriviridae.
A strain of this European type, called the "Lelystad virus" (LV) has been deposited with the Institut Pasteur, Paris, France under nr. I-1102, in connection with PCT WO 92/21375 by the Central Veterinary Institute, Lelystad, The Netherlands.
Another European strain has been described in EPA no. 91.202.646.5, and was deposited with the Collection Nationale de Cultures de Micro-organismes (CNCM) of the Institut Pasteur at Paris France under nr. I-1140.
This European strain has recently been described by Conzelmann et al. (Virology 193, 329-339, 1993).
The American type of the virus has been described by Benfield et al. (J. Vet. Diagn. Invest. 4, 127-133, 1992). A strain of the American type has been deposited with the ATCC under nr. VR-2332, and is mentioned in PCT WO 93/03760 and European Patent Application 0.529.584. An important observation was made by Wensvoort et al. (J. Vet. Diagn. Invest. 4, 134-138, 1992), early after the virus was found, when he compared American and European strains on the basis of their antigenic characteristics and demonstrated that the European and American strains display considerable antigenic differences.
The genomes of two European strains of PRRSV and parts of the genomes of US isolates have been molecularly cloned and sequenced (PCT WO 92/21375; Meulenberg et al., Virology 192, 62-72, 1993; Conzelmann et al., supra; Kwang et al., J. Vet. Diagn. Invest. 6, 293-296, 1994; Mardassi et al., J. Gen. Virol. 75, 681-685, 1994; Meng et al., Proc. 13th IPVS Congress, 61, 1994).
The RNA of the PRRSV genome comprises about 15 kb and 8 open reading frames (ORFs) can be identified therein, ORF 1A,B and ORFs 2 to 7. ORF 1A,B encodes a.o. the viral RNA polymerase, whereas the ORFs 2 to 6 encode the viral membrane (envelope) proteins. ORF 7 encodes the nucleocapsid protein. In infected cells a 3' coterminal nested set of six major subgenomic mRNAs can be demonstrated in both the European and US isolates (Meng et al., supra). Table 1 summarizes the characteristics of the ORFs encoding the structural proteins of PRRSV (Conzelmann et al., supra).
TABLE 1 ______________________________________ mRNA Calculated MW ORF (kb) Amino acids protein (kD) ______________________________________ 2 3.7 249 28.4 3 3.1 265 30.6 4 2.6 183 20.0 5 2.0 201 22.4 6 1.4 173 18.9 7 0.9 128 13.9 ______________________________________
The distribution of the ORFs on the PRRSV genome is shown in FIG. 1 and corresponds to that found by Meulenberg et al. (supra).
Little is known about the PRRSV proteins expressed by the ORFs mentioned before. 5 viral proteins of about 15-26 kD were identified in lysates of cells inoculated with an European or US PRRS isolate (Nelson et al., J. Clin. Microbiol. 31, 3184-3189, 1993 and Benfield et al, Proc. 13th IPVS Congress, 62, 1994). It was demonstrated that the ORF 7, ORF 6 and ORF 5 proteins are present in virions (Meulenberg et al., Virology 206, 155-163, 1995). It has been found now that two different forms, a small and a large form, of the ORF 3 and ORF 4 protein are present in cells infected with PRRSV, but only one form is present in purified virions isolated from the supernatant of infected cells. The two forms of each ORF protein are distinguished by the level of processing of their carbohydrate chains. The large forms, which are the forms present in the purified virions, are Endoglycosidase H (Endo-H) resistant, whereas the small forms are Endo-H sensitive. The substrate of the enzyme Endo-H is only the "high-mannosylated" form of the glycoproteins. This enzyme cleaves the oligosaccharide chain immediately after the carbohydrate group GlcNac which is coupled to the amino acid asparagin (Asn). More complex forms, i.e. forms in which the glycosylation process is complete, are resistant to Endo-H cleavage. The Endo-H resistance was used herein to determine the stage of the glycosylation process of the ORF 3 and 4 proteins and to distinguish the small ("high-mannose") forms of the ORF proteins from the large ("complex") form thereof. The enzyme Peptid-N-Glycohydrolase (PnGase-F) was used to obtain the completely deglycosalyted form (polypeptide backbone) of ORF 3 and 4. It was found that the ORF 3 and 4 glycoproteins incorporated in virons are the large ("complex") forms which resulted from their precursor small ("high mannose") forms by further processing, as a result of which said large forms became Endo-H resistant. The molecular weight (mw) as determined by SDS-PAGE of the ORF 3 and ORF 4 protein forms is shown in Table 2.
TABLE 2 ______________________________________ MW non-glycosylated MW small.sup.1 MW large.sup.2 ORF form (kD) form (kD) form (kD) ______________________________________ 3 26 41-44 55-60 4 15,5 32-35 40-47 ______________________________________ .sup.1 EndoH sensitive .sup.2 EndoH resistant
After expression of individual ORFs in eukaryotic cells transformed with the individual ORFs it was found that, in contrast to virus infected cells, exclusively the small forms of the ORF 3 and ORF 4 proteins were produced. Since the individual proteins are not completely processed in eukaryotic cells, most likely the transport of the proteins from the endoplasmic reticulum to the medial cisternae of the golgi apparatus is not possible. Surprisingly, it has been found now that in order to obtain the large forms of the PRRSV ORF 3 and ORF 4 proteins, co-expression of ORF 2, ORF 3 and ORF 4 in one cell is required and sufficient. During the infection process, animals are confronted with the mature (large) forms of the proteins. Therefore, the present invention provides a process for the recombinant DNA preparation of the mature large forms of PRRSV ORF 3 and ORF 4 proteins.